SOP: Detection of Salmonella in Foods and Environmental Surface Samples Using the BAX System Salmonella Assay

GIFSQ — Global Institute for Food Safety & Quality

Principle

This method detects Salmonella species in foods and environmental surface samples to assess compliance with Sections 4 and 7 of the Food and Drugs Act and other relevant federal regulations. Positive results must be confirmed with a cultural method. The method has been validated for all foods and a variety of environmental surface samples, run on the BAX System Q7 and X5 instruments. This revised method replaces MFLP-29, dated September 2017.

The BAX System is a screening tool that uses the Polymerase Chain Reaction (PCR) to amplify a specific fragment of bacterial DNA unique to Salmonella, with fluorescent detection of the PCR product. After enrichment, sample preparation takes about 1 hour of user time, and the automated procedure delivers results about 4 hours later. The automated fluorescent detection allows closed-tube testing, minimizing carry-over contamination with amplified DNA.

Two versions of the assay can be run on the Q7 or X5 platforms. The newer X5 instrument uses a side read instead of a top read, so its PCR tubes are optically transparent rather than opaque; all other reagents are the same in the X5 kit as in the Q7 kit. The hot start version of the test kit, BAX System PCR Assay for Salmonella 2, is interchangeable with the original BAX System PCR Assay for Salmonella.

Apparatus

• BAX System Q7 Start-Up Package (Cat. No. ASY2018), including:
• Cycler/detector with verification plates
• Computer workstation with Microsoft Windows operating system, BAX system amplification, and printer
• Automated Thermal Block (cat. no. DMCH2023) or dry block heaters with thermometer inserts for lysis tubes
• Capping/decapping tools
• Pipettes for reagent and sample transfers (5 µL, 10 µL, 10–50 µL 8 channel, repeat pipettor)
• Cooling block with inserts for lysis tubes and PCR tubes
• PCR tube holders
• Lysis tubes with caps and rack
• Barrier or filter tips for pipettes
• Powder-free nitrile gloves
• BAX System Q7 User Guide
• BAX System X5 Start-Up Package (Cat. No. MCH2007), including:
• Cycler/detector
• Computer workstation with Microsoft Windows operating system, BAX system amplification, and printer
• Automated Thermal Block (cat. no. DMCH2023)
• Capping/decapping tools
• Pipettes for reagent and sample transfers (5 µL, 10 µL, 10–50 µL 8 channel, repeat pipettor)
• Cooling block with inserts for lysis tubes and PCR tubes
• PCR tube holders
• Lysis tubes with caps and rack
• Barrier or filter tips for pipettes
• Powder-free nitrile gloves
• BAX System X5 User Guide
• Stomacher for homogenizing food samples
• Incubators capable of maintaining 35, 37, 42 and 42.5 °C

Reagents

• BAX System PCR Assay for Salmonella and BAX System PCR Assay for Salmonella 2 (Cat. KIT2012/KIT2011; sufficient for 96 tests): PCR tubes with tablets, flat optical caps, 2 lysis buffer bottles (12 mL per bottle) and protease solution (400 µL per vial), used to prepare working lysis reagent
• BAX System X5 PCR Assay for Salmonella (Cat. KIT2025; sufficient for 64 tests): PCR tubes with tablets, flat optical caps, 2 lysis buffer bottles (12 mL per bottle) and protease solution (400 µL per vial), used to prepare working lysis reagent
• Enrichment and regrowth broths, by food type:
• Buffered Peptone Water (BPW)
• To prepare: dissolve 10.0 g of peptone, 5.0 g of sodium chloride (NaCl), 3.5 g of disodium phosphate and 1.5 g of potassium dihydrogen phosphate in 1.0 L of distilled water; final pH 7.2 ± 0.2. Dispense into appropriate glassware and autoclave at 121°C for 15 min.
• Lactose broth
• To prepare: dissolve 3.0 g of beef extract, 5.0 g of peptone and 5.0 g of lactose in 1.0 L of distilled water; final pH 6.9 ± 0.2. Dispense and autoclave at 121 °C for 15 min.
• Brain Heart Infusion broth (BHI)
• To prepare: basal medium — infusion from 200.0 g calf brain, infusion from 250.0 g beef heart, 10.0 g proteose peptone, 2.0 g dextrose, 5.0 g sodium chloride (NaCl) and 2.5 g sodium phosphate (Na₂HPO₄) per 1.0 L of distilled water; final pH 7.4 ± 0.2. Dissolve the ingredients in 1.0 L of distilled water, distribute as required (tubes or flasks), and sterilize at 121 °C for 15 min.
• Brilliant Green Water
• Reconstituted non-fat dry milk
• Tetrathionate Brilliant Green Broth (TBG)
• To prepare: suspend 5.0 g of peptone/proteose peptone, 1.0 g of bile salts, 10.0 g of calcium carbonate (CaCO₃) and 30.0 g of sodium thiosulphate (Na₂S₂O₃) in 1.0 L of distilled water (pH 8.4 ± 0.2) and heat to boiling; cool to 45–50 °C. Prepare the complete medium on the day of use: aseptically add 1.0 mL of 1% brilliant green solution and 20.0 mL of iodine solution per litre of basal medium (brilliant green solution: dissolve 1 g of brilliant green dye in 100 mL of distilled water; iodine solution: suspend 25.0 g of potassium iodide and 30.0 g of iodine in 100 mL of distilled water, without heating). Mix thoroughly and aseptically dispense; do not heat the medium after the addition of iodine.
• Rappaport-Vassiliadis Soya Peptone Broth (RVS)
• To prepare: suspend 7.2 g of sodium chloride (NaCl), 4.5 g of soya peptone, 1.26 g of potassium phosphate (monobasic) (KH₂PO₄), 0.18 g of potassium phosphate (dibasic) (K₂HPO₄), 13.58 g of magnesium chloride, anhydrous (MgCl₂) and 0.036 g of malachite green in 1.0 L of distilled water (pH 5.2 ± 0.2); heat gently to dissolve, dispense into tubes, and autoclave at 115 °C for 15 min.
• TSB with 0.5% w/v K₂SO₃
• To prepare: suspend 17.0 g tryptone, 3.0 g soya peptone, 2.5 g dextrose, 5.0 g sodium chloride (NaCl), 2.5 g dipotassium phosphate (K₂HPO₄) and 5.0 g of potassium sulphite (K₂SO₃) in 1.0 L of distilled water (pH 7.3 ± 0.2); heat to boiling with constant stirring to dissolve, dispense, and autoclave at 121 °C for 15 min.
• Brilliant green dye, 1.0% w/v
• To prepare: dissolve 1.0 g of brilliant green dye in distilled water and make up to 100 mL.
• Reconstituted Non-Fat Dry Milk, prepared as follows:
• Basal medium: 100 g instant non-fat dry milk and 1.0 L distilled water
• Complete medium: suspend the instant non-fat dry milk in 1.0 L of distilled water and stir to dissolve; autoclave at 121 °C for 15 min

Sample preparation

Handling of sample units

• In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated or frozen, depending on the nature of the product; thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death
• Analyze sample units as soon as possible after their receipt in the laboratory

Preparation of samples

• Agitate liquids or free-flowing materials until the contents are homogeneous; if the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit
• Compositing can only be performed provided that the enrichment conditions (primary and secondary enrichment broths, the temperature and the duration of incubation) are the same as MFHPB-20 or have been validated as indicated in the table below
• Analytical units may be combined up to a maximum of 375 g, while maintaining the 1 in 10 dilution; when analyzing composites, pre-warm the enrichment broth to 35 ± 1.0 °C (see Appendix M of Volume 1, the Supplement on Interpretation of Compositing, for more details)
• Follow MFLP-41 for collection and transportation of environmental surface samples
• Prepare samples according to the food category, as below; after the pre-enrichment and enrichment or regrowth steps, proceed to the lysis step of the Procedure

Dairy products

• Frozen dairy: place 25 g aseptically in 225 mL Brilliant Green Water; blend, stomach, or vortex as required for thorough mixing; adjust pH to 6.0–7.4; incubate at 35 °C for 22–26 h
• Enrichment: add 10 µL enriched sample to 500 µL BHI (room temperature); incubate at 37 °C for 3 h; compositing not allowed
• Heat processed dairy (Option A): place 25 g aseptically in 225 mL BPW; blend, stomach, or vortex as required; adjust pH to 6.0–7.4; incubate at 35 °C for 22–26 h
• Enrichment: add 10 µL enriched sample to 500 µL BHI (room temperature); incubate at 37 °C for 3 h; compositing not allowed
• Heat processed dairy (Option B): place 25 g aseptically in 225 mL BPW; blend, stomach, or vortex as required; adjust pH to 6.0–7.4; incubate at 35 °C for 18–24 h
• Enrichment: transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix; compositing allowed
• Raw dairy: place 25 g aseptically in 225 mL BPW; blend, stomach, or vortex as required; adjust pH to 6.0–7.4; incubate raw milk cheese at 42 °C and other raw dairy products at 35 °C, for 18–24 h
• Enrichment: transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix; compositing allowed

Fish and seafood

• Raw fish and seafood (Option A): place 25 g sample aseptically in 225 mL lactose broth; blend, stomach, or vortex as required; let stand at room temperature for 55–65 minutes; adjust pH to 6.8 ± 0.2; incubate at 35 °C for 22–26 h
• Enrichment: not required; compositing not allowed
• Raw fish and seafood (Option B): place 25 g sample aseptically in 225 mL BPW; blend, stomach, or vortex as required; adjust pH to 6.0–7.4; incubate at 35 °C for 18–24 h
• Enrichment: transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix; compositing allowed
• Heat-processed fish and seafood (Option A): place 25 g sample aseptically in 225 mL lactose broth; blend, stomach, or vortex as required; let stand at room temperature for 55–65 minutes; adjust pH to 6.8 ± 0.2; incubate at 35 °C for 22–26 h
• Enrichment: add 10 µL enriched sample to 500 µL BHI (room temperature); incubate at 37 °C for 3 h; compositing not allowed
• Heat-processed fish and seafood (Option B): place 25 g sample aseptically in 225 mL BPW; blend, stomach, or vortex as required; adjust pH to 6.0–7.4; incubate at 35 °C for 18–24 h
• Enrichment: transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix; compositing allowed
• Frozen fish and seafood (Option A): place 25 g sample aseptically in 225 mL BPW; blend, stomach, or vortex as required; adjust pH to 6.0–7.4; incubate at 35 °C for 22–26 h
• Enrichment: not required; compositing not allowed
• Frozen fish and seafood (Option B): place 25 g sample aseptically in 225 mL BPW; blend, stomach, or vortex as required; adjust pH to 6.0–7.4; incubate at 35 °C for 18–24 h
• Enrichment: transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix; compositing allowed

Chocolate and bakery products

• Dry (powder) chocolate/bakery products (Option A): place 25 g sample aseptically with 225 mL of reconstituted non-fat dried milk medium; blend, stomach, or vortex as required; let stand at room temperature for 55–65 min; adjust pH to 6.8 ± 0.2; add 0.45 mL brilliant green dye (1.0% w/v); incubate at 35 °C for 22–26 h
• Enrichment: add 10 µL enriched sample to 500 µL BHI (room temperature); incubate at 37 °C for 3 h; compositing allowed
• Dry (powder) chocolate/bakery products (Option B): place 25 g sample aseptically with 225 mL of reconstituted non-fat dried milk medium; blend, stomach, or vortex as required; let stand at room temperature for 55–65 min; adjust pH to 6.8 ± 0.2; add 0.45 mL brilliant green dye (1.0% w/v); incubate at 35 °C for 18–24 h
• Enrichment: transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix; compositing allowed
• Low-moisture chocolate/bakery products (Option A): place 25 g sample aseptically with 225 mL of reconstituted non-fat dried milk medium; blend, stomach, or vortex as required; let stand at room temperature for 55–65 min; adjust pH to 6.8 ± 0.2; add 0.45 mL brilliant green dye (1.0% w/v); incubate at 35 °C for 22–26 h
• Enrichment: add 10 µL enriched sample to 500 µL BHI (room temperature); incubate at 37 °C for 3 h; compositing allowed
• Low-moisture chocolate/bakery products (Option B): place 25 g sample aseptically with 225 mL of reconstituted non-fat dried milk medium; blend, stomach, or vortex as required; let stand at room temperature for 55–65 min; adjust pH to 6.8 ± 0.2; add 0.45 mL brilliant green dye (1.0% w/v); incubate at 35 °C for 18–24 h
• Enrichment: transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix; compositing allowed

Fruit and vegetable-based products

• Nuts and nut meats (includes in shell, shelled, seeds and nut/seed butters and spreads): place 25 g sample aseptically in 225 mL lactose broth; blend, stomach, or vortex as required; let stand at room temperature for 55–65 min; adjust pH to 6.8 ± 0.2; incubate at 35 °C for 22–26 h
• Enrichment: add 10 µL enriched sample to 500 µL BHI (room temperature); incubate at 37 °C for 3 h; compositing allowed
• Optional procedure for nut/seed butters and spreads: place 25 g sample aseptically in 225 mL BPW; blend, stomach, or vortex as required; adjust pH to 6.0–7.4; incubate at 35 °C for 18–24 h
• Enrichment: transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix; compositing allowed
• Whole cantaloupes: place the entire cantaloupe aseptically into a stomacher bag and add up to 1.5 times the weight of the cantaloupe of BPW; when the analysis is done on a larger cantaloupe (about 2 kg), a lesser volume of BPW may be used provided there is sufficient volume to totally submerge the fruit; mix for 30 sec by manually rubbing the rind; submerge and hold at room temperature for 30 min and again manually rub the rind; adjust pH to 6.0–7.4; incubate at 35 °C for 20–24 h, adding a weight to the outside of the stomacher bag to ensure that the fruit remains submerged during incubation
• Enrichment: transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix; compositing does not apply

Raw poultry products

• Raw processed poultry (Option A): place 25 g sample aseptically in 225 mL BPW; blend, stomach, or vortex as required; adjust pH to 6.0–7.4; incubate at 42 °C for 24–26 h
• Enrichment: not required; compositing not allowed
• Frozen poultry, raw or raw processed (Option B): place 25 g sample aseptically in 225 mL BPW; blend, stomach, or vortex as required; adjust pH to 6.0–7.4; incubate at 35 °C for 18–24 h
• Enrichment: transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix; compositing allowed

Spices

• Follow the dilution ratio guidance in MFHPB-20 using a 1 in 10 (and if applicable, higher) dilution scheme (as in, 25 g sample in 225 mL broth); for some spices, multiple primary enrichments are required to encompass different dilution ratios
• Enrichment: transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively, and repeat if multiple dilutions were used; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix — if multiple dilutions were used, combine 2 mL from each RVS and TBG corresponding to a particular dilution separately; compositing allowed

Environmental surface samples

• For environmental sponges, add to 100 mL of BPW, or composite up to 10 sponges with 100 mL of BPW per sponge, and mix well by hand; for environmental swabs, place the swab into a tube with 10 mL of BPW, or composite up to 10 swabs with 10 mL of BPW per swab, and mix well by hand; incubate the sample in BPW at 35 °C for 18–24 h
• Enrichment: not required; an optional regrowth step may be useful for indeterminate results — add 10 µL enriched sample to 500 µL BHI (room temperature); incubate at 37 °C for 3 h; compositing allowed

Other food categories

• Option A: place 25 g sample aseptically in 225 mL of an appropriate pre-enrichment broth for the food type according to the specifications in MFHPB-20 (in cases where MFHPB-20 provides the option of nutrient broth or BPW, BPW is to be used); blend, stomach, or vortex as required for thorough mixing and adjust pH as indicated in MFHPB-20; incubate at 35 °C for 22–26 h
• Enrichment: add 10 µL enriched sample to 500 µL BHI (room temperature); incubate at 37 °C for 3 h; compositing not allowed
• Option B: place 25 g sample aseptically in 225 mL of an appropriate pre-enrichment broth for the food type according to the specifications in MFHPB-20 (in cases where MFHPB-20 provides the option of nutrient broth or BPW, BPW is to be used); blend, stomach, or vortex as required for thorough mixing and adjust pH as indicated in MFHPB-20; incubate at 35 °C for 18–24 h
• Enrichment: for all other foods, except for oatmeal products, transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of RVS and TBG broths, respectively; incubate for 24 ± 2 h at 42.5 °C; combine 2 mL from each broth and mix; compositing allowed; Option A must be used for oatmeal products

Procedure

Prepare the equipment

• Make sure the cooling blocks have been refrigerated overnight
• Warm up the heating blocks; check that temperatures are set to 37 °C and 95 °C
• Initialize the BAX System cycler/detector
• Create a rack file
  ↓
• Select RUN FULL PROCESS in the menu bar to warm up the cycler/detector prior to placing the samples into the system

Perform sample lysis

• Arrange the required number of lysis tubes (one for each sample and one for the blank) in the rack according to the rack file; prepare blank, positive and negative controls as per the laboratory’s requirements
• Prepare the lysis reagent by pipetting 150 µL of protease into one 12 mL bottle of lysis buffer
• Add 200 µL of lysis reagent to each lysis tube
• After the appropriate pre-enrichment, and enrichment or regrowth step, transfer 5 µL of enriched sample to the corresponding lysis tube
• Secure the caps; when using manual heating blocks, heat the tubes at 37 °C for 20 minutes, then at 95 °C for 10 minutes; if using the Automated Thermal Block, use the preset parameters, which include cooling of the samples
  ↓
• Place the lysis tubes in the cooling block for at least 5 min

Hydrate the PCR tablets with the lysates

• Place the PCR tube holder onto the PCR cooling block
• Arrange one PCR tube per sample in the holder
• Remove and discard the lid from one strip of tubes at a time
• Using a multi-channel pipette, transfer 50 µL of each lysed sample into a corresponding PCR tube
• Cover the tubes with a new optical cap strip and secure tightly; repeat for all samples
  ↓
• Take the entire cooling block to the cycler/detector; the samples should remain in the cooling block until the cycler/detector is ready for loading, but no more than 30 min after tablet hydration

Amplify and detect

• Follow the screen prompts of the PCR Wizard to load the samples, run the program and unload the samples, as specified in the BAX System Q7 and X5 User Guides

Review the results

• Read the result display: a green minus sign means a negative result; a red plus sign means a positive result; a yellow question mark means an indeterminate result; a yellow question mark with a red bar means an error
  ↓
• For indeterminate results, repeat the analysis beginning with the lysis transfer step

Confirm positive results

• Using the incubated enrichment broth, proceed with confirmation as follows:
• If there was no regrowth step, or a regrowth was performed in BHI: transfer an aliquot of the primary enrichment broth to the selective enrichment broths described in MFHPB-20; continue with MFHPB-20 for confirmation
• If there was a selective enrichment in TBG and RVS: proceed with the plating and confirmation steps as described in the “selective plating” section of MFHPB-20, plating each of the RVS and TBG secondary enrichments separately

Interpretation of results

• Negative results can be reported as Salmonella Not Detected
• Repeated indeterminate (that is, more than one) or error results must be treated as presumptive positive; proceed with confirmation as described above

Precautions

• The laboratory supervisor must ensure that completion of the analysis described in this method is done in accordance with the International Standards reference ISO/IEC 17025 (latest version): “General Requirements for the Competence of Testing and Calibration Laboratories”
• The temperatures of the incubators or water baths must be maintained at the recommended temperatures; the following applies to the steps of the method which apply to growth only — where a temperature of 37 °C or lower is recommended, the temperature may be ± 1.0 °C (for example, 35 ± 1.0 °C), but where higher temperatures are recommended, the incubators or water baths must be maintained within 0.5 °C, due to potential lethality of the higher temperatures on the microorganism being isolated

References

• Health Canada. Detection of Salmonella in Foods and Environmental Surface Samples Using the BAX System Salmonella Assay. Compendium of Analytical Methods, Volume 3, Laboratory Procedure MFLP-29, May 2020 (version 2). Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada.
• Reid, A. 2009. MFHPB-20. Methods for the Isolation and Identification of Salmonella from Foods. In: Volume 2. Compendium of Analytical Methods, Ottawa.
• Health Canada. Appendix M: Supplement to All Bacterial Qualitative Methods in the Compendium of Analytical Methods: Interpretation of Compositing. In: Volume 1. Compendium of Analytical Methods, Ottawa.